Circ_0138960 contributes to lipopolysaccharide‐induced periodontal ligament cell dysfunction

Abstract Background Periodontitis is a common oral inflammatory disease, and lipopolysaccharide (LPS) is a key risk factor in periodontitis pathology. Here, we used LPS‐induced periodontal ligament cells (PDLCs) to explore the molecular mechanism of periodontitis. Methods Cell viability, proliferation, and apoptosis were analyzed by Cell Counting Kit‐8, 5‐ethynyl‐20‐deoxyuridine (EDU), and flow cytometry assays, respectively. Apart from that, their targeting relationship was validated using dual‐luciferase reporter and RNA‐pull down. Results Circular RNA_0138960 (circ_0138960) was notably upregulated in periodontitis sufferers (p < .001) and LPS‐disposed PDLCs (p < .05). LPS exposure dampened PDLC proliferation, and promoted apoptosis and inflammation (p < .05). Circ_0138960 acted as a microRNA sponge for miR‐518a‐5p to affect histone deacetylase 6 (HDAC6) expression. Circ_0138960 absence‐mediated protective effects in LPS‐induced PDLCs were largely abrogated via silencing miR‐518a‐5p or HDAC6 overexpression (p < .05). Conclusion Circ_0138960 promoted LPS‐induced dysfunction in PDLCs by targeting miR‐518a‐5p/HDAC6 axis, which provided novel potential therapeutic targets for periodontitis.


| INTRODUCTION
As a progressive disease of periodontal tissue, Periodontitis is ascribed to the accumulation of subgingival pathogens. 1 The pathology of periodontitis is associated with environmental factors (bad living habits and smoking) and genetic factors. 2 Current research revealed that clarification on the mechanisms involving periodontitis is of great significance for the prevention of periodontitis. Lipopolysaccharide (LPS) is highly toxic to the periodontal ligament and can induce cellular dysfunction, thus contributing to periodontitis progression. 3 Hence, LPS-induced periodontal ligament cells (PDLCs) are generally utilized as periodontitis cell models in vitro. 4,5 Circular RNAs (circRNAs) with conserved stable loop structures highlighted their potential as biomarkers for human diseases. 6 CircRNAs play important regulatory roles in periodontitis pathology. 5,7 Circ_0081572 expression is downregulated in patients with periodontal disease, and circ_0081572 overexpression attenuates LPS-induced dysfunction in PDLCs. 8 Here, we focus on an abnormally upregulated circRNA, circ_0138960, in patients with periodontal disease, 9 but its biological role and mechanism in periodontitis pathology remain to be clarified.
Recent reports have described that circRNAs act as microRNA (miRNA) sponges to indirectly modulate gene expression, thus affecting cell behaviors. 10,11 This study first investigated the role of circ_0138960 in LPStriggered PDLC dysfunction. Subsequently, we explored the downstream miRNA/Messenger RNA (mRNA) axis of circ_0138960 to uncover its working mechanism in periodontitis pathology.

| Gingival tissues
After being signed written informed consent, human gingival tissues were collected from patients with periodontal disease (n = 37) or from normal controls (n = 25) who received gingivectomy during orthodontic treatment at Dongying Shengli Oilfield Central Hospital. Diagnostic criteria of periodontitis were presented as follows: (1) redness and swelling of the gingiva on the surface of the periodontal pocket or bleeding after probing; (2) probe depth > 3 mm and attachment loss > 1 mm; (3) X-ray showed horizontal or vertical resorption of alveolar bone. Exclusion criteria included patients with systemic diseases and antibiotic intake within 1 month before the surgery. Approval to perform this project was acquired from the Ethics Committee of Dongying Shengli Oilfield Central Hospital. NO. S2020623. Table 1 displayed the clinicopathologic characteristics of periodontitis sufferers.
2.2 | Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) RNA samples were isolated from gingival tissues and PDLCs with Trizol reagent (Invitrogen). RNA was reversely transcribed to complementary DNA (cDNA) using an M-MLV kit (Invitrogen) and miRNA cDNA Synthesis Kit (GeneCopoeia). Then, qPCR was carried out using the Power SYBR-Green PCR master mix (Applied Biosystems). After normalization with β-actin or U6, the obtained results were processed using the 2 C −ΔΔ t . All primers were shown in Table 2

| LPS treatment
PDLCs were stimulated with Porphyromonas gingivalis LPS (Sigma) at the dose of 2.5, 5, or 10 ng/μl for 12 h. Finally, PDLCs were treated with 10 ng/μl LPS for 12 h to induce the periodontitis-like injury, which was used for further analysis.

| Dual-luciferase reporter assay
A partial sequence of circ_0138960 or HDAC6 3' untranslated region (UTR) was amplified by qPCR. Meanwhile, their mutant sequence was synthesized via site-directed mutation. The constructed reporter plasmids were termed circ_0138960 WT/MUT and HDAC6-3'UTR WT/MUT . PDLCs were cotransfected with small RNAs and constructed plasmids, followed by analysis according to Dual-Luciferase Reporter Assay kit (Promega) after transfection for 24 h.

| Statistical analysis
The mean between groups was compared using Student's t-test or one-way analysis of variance followed by Tukey's test. GraphPad Prism 7.0 software (GraphPad) was used for statistical analysis. p < .05 was regarded as the threshold of significance.

| Circ_0138960 expression is elevated in patients with periodontal disease
Circ_0138960 (366 nt) is derived from the back-splicing of the gene GDA ( Figure 1A). Meanwhile, our data displayed that circ_0138960 was only amplified using divergent primers in the cDNA group, rather than gDNA ( Figure 1B). The expression of circ_0138960 in periodontitis was analyzed. Circ_0138960 was notably upregulated in patients with periodontal disease (n = 37) versus normal controls (n = 25) ( Figure 1C, p < .001). To identify the association of circ_0138960 expression with the clinicopathologic characteristics, the 37 patients with periodontitis disease were then classified in Table 1.
Result displayed that circ_0138960 expression was associated with Order of severity and Grade of periodontitis (p < .05). Overall, circ_0138960 was associated with periodontitis pathology.

| HDAC6 overexpression reverses circ_0138960 interference-mediated impacts in PDLCs upon LPS treatment
We analyzed the biological role of HDAC6 in LPS-induced PDLCs by loss-of-function experiments. Knockdown efficiency of si-HDAC6 in PDLCs was validated (Supporting Information: Figure 2A). LPS-induced PDLC dysfunction was effectively diminished via silencing HDAC6 (Supporting Information: Figure 2B-2Q), indicating that HDAC6 knockdown protected PDLCs from LPS-induced dysfunction. Western blot assay confirmed the overexpression efficiency of HDAC6 plasmid in PDLCs ( Figure 7A). The protective action of cell viability and proliferation of LPSinduced PDLCs caused by circ_0138960 depletion was reversed by the overexpression of HDAC6 ( Figure 7B−G). Circ_0138960 silencing suppressed LPS-induced apoptosis in PDLCs, and the addition of HDAC6 plasmid triggered cell apoptosis again ( Figure 7H−L). The addition of HDAC6 plasmid also induced the inflammation of PDLCs ( Figure 7M−P). Taken together, a protective role of circ_0138960 absence on LPS-induced PDLCs was partly mitigated via HDAC6 downregulating.

| DISCUSSION
Inflammation is considered to be a major factor in periodontitis pathology. Therefore, alleviating inflammation-induced PDLCs injury is important for the treatment of periodontitis. 20 LPS is widely utilized in F I G U R E 4 Circ_0138960 acts as a molecular sponge for miR-518a-5p. (A) Circinteractome database showed that circ_0138960 harbored the potential binding sites with miR-518a-5p. (B) RT-qPCR was conducted to determine the expression of miR-518a-5p in the gingival tissues of patients with periodontal disease (n = 37) and normal controls (n = 25). (C) RT-qPCR was conducted to examine the level of miR-518a-5p in PDLCs stimulated with LPS at the dose of 2.5, 5, or 10 ng/μl for 12 h. (D and E) The target relation between circ_0138960 and miR-518a-5p was confirmed by dual-luciferase reporter assay and RNA-pull down assay. *p < .05, **p < .01, ***p < .001. LPS, lipopolysaccharide; PDLCs, periodontal ligament cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
inflammation-associated cell models in vitro. 21 In this study, LPS-induced PDLCs were used as a periodontitis cell model in vitro. Our study confirmed that LPS stimulation reduced PDLC proliferation and induced apoptosis and inflammation in a dose-dependent manner. Noncoding RNAs, including circRNAs, are identified as promising targets for inflammationassociated diseases such as periodontitis. 7,22 Here, we found that circ_0138960 was upregulated in patients with periodontal disease, which was consistent with a previous study. 9 LPS exposure gradually upregulated circ_0138960 content in PDLCs. In addition, we found that circ_0138960 depletion relieved LPS-induced PDLC dysfunction.
Accumulating evidence has suggested that circRNAs play important regulatory roles in multiple human diseases by acting as miRNA sponges. 23,24 For instance, circ_0003204 restrains endothelial cell growth, migration, and angiogenesis in atherosclerosis by sponging miR-370-3p. 25 Serving as a miR-31-5p sponge, circ-BPTF might aggravate bladder cancer development. 26 Using bioinformatics analysis, miR-518a-5p appeared as a circ_0138960 target in PDLCs. miR-518a-5p expression was significantly reduced in patients with periodontal disease. Meanwhile, we uncovered that LPS treatment decreased the miR-518a-5p level in PDLCs in a dosedependent manner. A previous study reported that lncRNA 01126 contributes to periodontitis pathogenesis by sponging miR-518a-5p, 27 indicating that miR-518a-5p played a protective role in periodontitis. We found miR-518a-5p downregulation might reverse circ_0138960 absenceprovoked protective action in LPS-stimulated PDLCs, indicating that circ_0138960 silencing protected PDLCs against LPS-caused damage largely via miR-518a-5p. F I G U R E 6 HDAC6 is a target of miR-518a-5p. (A) Bioinformatics database targetscan predicted the possible binding sequence between miR-518a-5p and HDAC6. (B and C) The mRNA and protein levels of HDAC6 were determined in the gingival tissues of patients with periodontal disease and normal controls by RT-qPCR and Western blot assay. (D) Western blot assay was conducted to measure the protein level of HDAC6 in PDLCs stimulated with LPS at the dose of 2.5, 5, or 10 ng/μl for 12 h. (E) Their binding was verified by dual-luciferase reporter assay. (F) Western blot assay was performed to determine the protein level of HDAC6 in PDLCs transfected with anti-miR-NC or anti-miR-518a-5p. (G) Western blot analysis of effects of si-circ_0138960 and anti-miR-518a-5p on HDAC6 protein level. *p < .05, **p < .01, ***p < .001. HDAC6, histone deacetylase 6; mRNA, messenger RNA; PDLCs, periodontal ligament cells; RT-qPCR, reverse transcriptionquantitative polymerase chain reaction.
It is well established that miRNAs can accomplish their biological function via interacting with mRNAs. 28,29 Our findings uncovered that miR-518a-5p is directly bound to the 3'UTR of HDAC6 in PDLCs. HDAC6 is a member of the histone deacetylase family, and it regulates multiple cellular processes through its histone deacetylase activity. 30 A previous study found that IL-1β elevates the mRNA expression of HDAC6 in periodontal ligament fibroblasts, 16 implying its pivotal role in periodontitis pathology. We confirmed that HDAC6 was elevated in periodontitis sufferers and LPSstimulated PDLCs. In addition, we uncovered circ_0138960 positively modulated HDAC6 content via sponging miR-518a-5p in PDLCs. HDAC6 knockdown protected PDLCs from LPS-induced dysfunction. Beyond that, compensation experiments disclosed that circ_0138960 silencing-induced protective influences in LPS-stimulated PDLCs were largely offset by HDAC6 accumulation, suggesting that circ_0138960 interference played protective impacts in LPS-stimulated PDLCs largely by downregulating HDAC6.
In summary, our findings confirmed that circ_0138960 contributed to LPS-induced dysfunction in PDLCs by miR-518a-5p/HDAC6 (Figure 8). This new ceRNA mechanism provides novel potential therapeutic targets for periodontitis. However, the current research is limited by the small sample size and the lack of animal model experiments. Hence, our future research will try to include more patients and construct periodontitis animal models to further verify our conclusions. Furthermore, the working mechanism of HDAC6 in periodontitis pathology still needs to be explored.